TRAIL及其受体与隐球菌性脑膜炎的关联性研究

目的研究建立实时荧光定量（RFQ）-PCR法测定TRAIL及其受体DeR1、DcR2、DR4、DR5 mRNA含量的方法，探讨其在隐球菌性脑膜炎患者外周血单个核细胞中表达的检测价值，并分析隐球菌性脑膜炎患者细胞因子与TRAIL及其受体的相关性。方法设计特异性的引物和探针，以人基因GAPDH为内参照，用实时定量PCR的方法检测35例健康人和35例隐球菌性脑膜炎患者外周血单个核细胞中TRAIL及其受体mRNA的表达水平，采用ELISA方法检测血清IL-4、IL-10、TNF-α、IFN-γ浓度。结果与健康人比较，隐球菌性脑膜炎患者外周血单个核细胞中TRAIL mRNA的表达明显下降（P〈0．01）；受体DeR1 mRNA的表达显著升高（P〈0．01），受体DeR2、DR4、DR5 mRNA的变化无统计学意义。隐脑患者血清中IL-10显著升高（P〈0．01），与TRAIL的变化成负相关；IFN-γ显著下降（P〈0．01），与TRAIL的变化成正相关。结论隐球菌性脑膜炎患者TRAIL下降及其受体DeR1mRNA的表达明显升高，提示TRAIL及其受体DeR1可能参与隐球菌性脑膜炎的疾病进程，这为有效治疗隐球菌性脑膜炎提供了新的线索。     

YM155 sensitizes triple‐negative breast cancer to membrane‐bound TRAIL through p38 MAPK‐ and CHOP‐mediated DR5 upregulation

Because available treatments have limited efficacy in triple‐negative breast cancer (TNBC), the identification of new therapeutic strategies to improve patients' outcome is urgently needed. In our study, we investigated the effects of the administration of the small molecule selective survivin suppressant YM155, alone or in association with CD34+ cells transduced with a replication‐deficient adenovirus encoding the human tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) gene (CD34‐TRAIL+ cells), in three TNBC cell models. YM155 exposure significantly impaired TNBC cell growth and selectively modulated survivin expression at both mRNA and protein level. In addition, co‐culturing YM155‐treated TNBC cells with CD34‐TRAIL+ cells resulted in markedly increased cytotoxic effect and apoptotic response in comparison with single treatments. Such a chemosensitizing effect was observed only in TNBC cells inherently expressing DR5 and relied on the ability of YM155 to upregulate DR5 expression through a p38 MAPK‐ and CHOP‐dependent mechanism. YM155/CD34‐TRAIL+ combination also showed a significant inhibitory effect on the growth of DR5‐expressing TNBC cells following xenotransplantation into NOD/SCID mice, in the absence of toxicity. Overall, our data (i) provide, for the first time, evidence that YM155 sensitizes TNBC cells to CD34‐TRAIL+ cells‐induced apoptosis by a mechanism involving the downregulation of survivin and the simultaneous p38 MAPK‐ and CHOP‐mediated upregulation of DR5, and (ii) suggest the combination of YM155 with TRAIL‐armed CD34+ progenitor cells as a promising therapeutic option for patients with TNBC expressing DR5.     

Engineered adenoviruses combine enhanced oncolysis with improved virus production by mesenchymal stromal carrier cells

Oncolytic viruses have demonstrated in pre‐clinical and clinical studies safety and a unique pleiotropic activity profile of tumor destruction. Yet, their delivery suffers from virus inactivation by blood components and sequestration to healthy tissues. Therefore, mesenchymal stromal cells (MSCs) have been applied as carrier cells for shielded virus delivery to tumors after ex vivo infection with oncolytic viruses. However, infection and particle production by MSCs have remained unsatisfying. Here, we report engineered oncolytic adenoviruses (OAds) for improved virus production and delivery by MSCs. OAds are uniquely amenable to molecular engineering, which has facilitated improved tumor cell destruction. But for MSC‐mediated regimens, OAd engineering needs to achieve efficient infection and replication in both MSCs and tumor cells. We show that an Ad5/3 chimeric OAd capsid, containing the adenovirus serotype 3 cell‐binding domain, strongly increases the entry into human bone marrow‐derived MSCs and into established and primary pancreatic cancer cells. Further, we reveal that OAd with engineered post‐entry functions—by deletion of the anti‐apoptotic viral gene E1B19K or expression of the death ligand TRAIL—markedly increased virus titers released from MSCs, while MSC migration was not hampered. Finally, these virus modifications, or viral expression of FCU1 for local 5‐FC prodrug activation, improved tumor cell killing implementing complementary cytotoxicity profiles in a panel of pancreatic cancer cell cultures. Together, our study establishes post‐entry modification of OAd replication for improving virus delivery by carrier cells and suggests a panel of optimized OAds for future clinical development in personalized treatment of pancreatic cancer.     

Relationship between tumour necrosis factor‐related apoptosis inducing ligand (TRAIL) and vascular endothelial growth factor in human multiple myeloma patients

Tumour necrosis factor‐alfa (TNF‐α) is an inflammatory cytokine with a wide spectrum of biological activity, including angiogenesis. Tumour necrosis factor‐related apoptosis inducing ligand (TRAIL), which belongs to the TNF family of proteins, plays a role in the regulation of vascular responses, but its effect on the formation of new blood vessels (angiogenesis) is unclear. We analysed TRAIL concentrations in parallel with pro‐angiogenic cytokines in serum and their expression in trephine biopsy (TB) in 56 patients with newly diagnosed IgG MM and 24 healthy volunteers. The study showed statistically higher concentrations of TRAIL and TNF‐α, as well as of VEGF and its receptor, in MM patients compared to healthy volunteers and patients in advanced stages of the disease. Furthermore, we observed a significant decrease in all studied pro‐angiogenic cytokines and significant increase of TRAIL concentration after anti‐angiogenic therapy, with meaningful differences between responders (at least partial remission) and patients with progression during the induction treatment. It was also established that TRAIL correlated statistically and negatively with pro‐angiogenic cytokines such as VEGF with its receptor and expression of VEGF and syndecan‐1 in TB. In summary, our data indicate that in MM patients, both clinical course and treatment responsiveness are associated with dynamic yet corresponding changes of levels of TRAIL parallel pro‐angiogenic mediators such as VEGF with its receptor and expression of VEGF and syndecan‐1 in TB. Copyright © 2014 John Wiley & Sons, Ltd.     

FTY720 enhances TRAIL-mediated apoptosis by up-regulating DR5 and down-regulating Mcl-1 in cancer cells

FTY720, Fingolimod, is a functional antagonist to the sphingosine-1-phoaphate (S1P) receptor and an inhibitor of sphingosine kinase 1. Here, we showed that a combination of FTY720 and TRAIL induced apoptosis in human renal, breast, and colon carcinoma cells. Most importantly, this combination had no effect on normal cells. Furthermore, the combined treatment with FTY720 and TRAIL reduced tumor growth in xenograft models. FTY720 up-regulated death receptor (DR)5 at post-translational level. Knockdown of DR5 markedly blocked apoptosis induced by the combined treatment. FTY720 also inhibited Mcl-1 expression at the post-translational level. Over-expression of Mcl-1 blocked apoptosis induced by FTY720 and TRAIL. Interestingly, phospho-FTY720 and inhibitors of sphingosine kinase failed to enhance TRAIL-induced apoptosis. Thus, FTY720 enables TRAIL-induced apoptosis through up-regulation of DR5 and down-regulation of Mcl-1 in human cancer cells.     

Delphinidin sensitizes prostate cancer cells to TRAIL-induced apoptosis,by inducing DR5 and causing caspase-mediated HDAC3 cleavage

TRAIL can induce apoptosis in some cancer cells and is an immune effector in the surveillance and elimination of developing tumors. Yes, some cancers are resistant to TRAIL. Delphinidin, a polyphenolic compound contained in brightly colored fruits and vegetables, has anti-inflammatory, anti-oxidant, and anti-tumorigenic activities. Here we showed that delphinidin sensitized TRAIL-resistant human prostate cancer cells to undergo apoptosis. Cells treated with delphinidin and TRAIL activated the extrinsic and intrinsic pathways of caspase activation. TRAIL-induced apoptosis in prostate cancer cells pretreated with delphinidin was dependent on death receptor 5 (DR5) and downstream cleavage of histone deacetylase 3 (HDAC3). In conclusion, delphinidin sensitizes prostate cancer cells to TRAIL-induced apoptosis by inducing DR5, thus causing caspase-mediated HDAC3 cleavage. Our data reveal a potential way of chemoprevention of prostate cancer by enabling TRAIL-mediated apoptosis.     

Caspase-8和Bcl-2在脑肿瘤干细胞肿瘤坏死因子相关凋亡诱导配体耐药中的作用研究

 目的 探讨肿瘤坏死因子相关凋亡诱导配体(TRAIL)对脑肿瘤干细胞生长的影响,研究Caspase-8和Bcl-2在肿瘤坏死因子相关凋亡诱导配体耐药中的作用。方法 应用CD133免疫磁珠分选法获得脑肿瘤干细胞,流式细胞术检测分选阳性率;亚神经球形成实验分析脑肿瘤干细胞的自我更新能力和增殖能力;四甲基偶氮唑盐法检测肿瘤坏死因子相关凋亡诱导配体对脑肿瘤干细胞生长的影响;Western blot法检测DR5、FADD、Caspase-8和Bcl-2蛋白的表达。结果 分选后CD133⁺细胞即脑肿瘤干细胞可达71%;脑肿瘤干细胞以神经球的方式生长;肿瘤坏死因子相关凋亡诱导配体作用前后,脑肿瘤干细胞都有极强的形成神经球的能力;肿瘤坏死因子相关凋亡诱导配体可以引起脑肿瘤干细胞死亡,加入Caspase-8或Caspases抑制剂,可明显阻断肿瘤坏死因子相关凋亡诱导配体所引起的细胞死亡(P<0.05);Western blot结果显示,脑肿瘤干细胞表达Caspase-8 蛋白水平降低,表达Bcl-2蛋白水平增加(P<0.05)。结论 脑肿瘤干细胞是脑肿瘤对肿瘤坏死因子相关凋亡诱导配体耐药的根源,Caspase-8蛋白表达减少及Bcl-2蛋白表达增强使Caspase-8不能活化,导致了脑肿瘤干细胞发生肿瘤坏死因子相关凋亡诱导配体耐药。     

XIAP siRNA、Embelin对TRAIL诱导肝癌细胞生长抑制和细胞凋亡的影响

目的 研究X-连锁凋亡抑制蛋白(X-linked inhibitor of apoptosis,XIAP) siRNA或XIAP拮抗剂Embelin对肿瘤坏死因子相关凋亡诱导配体(tumor necrosis factor-related apoptosis-inducing ligand,TRAIL)所致肝癌细胞HepG2生长抑制和细胞凋亡的影响.方法 HepG2细胞转染XIAP siRNA或者阴性对照,然后给予TRAIL处理.此外,HepG2细胞给予TRAIL、Embelin或者联合处理.XIAP表达水平变化、生长抑制、caspase-3活性分别用Western blot、MTT测定、caspase-3荧光法检测.切割PARP的表达水平用Western blot测定.结果 XIAP蛋白表达水平在转染XIAP siRNA后显著下调.与阴性对照相比,XIAP siRNA显著增强TRAIL在100 ng/ml(6.8％±1.2％比11.8％±4.0％,P＜0.05)和1000 ng/ml(18.9％±2.0％比26.6％±1.5％,P＜0.01)的生长抑制作用.在转染XIAP siRNA后,TRAIL诱导caspase-3的活性和PARP的切割显著增强.此外,Embelin显著增强TRAIL对HepG2细胞的生长抑制(P＜0.01)、caspase-3活化(P＜0.01)和PAPR切割.结论 XIAP siRNA或Embelin在肝细胞癌的临床治疗方面具有潜在应用前景.     

膀胱癌中DR4和DR5的表达水平与患者术后化疗及患者预后的关系

目的：在临床前期实验中,肿瘤坏死因子相关凋亡诱导配体（TRAIL）与化疗药物在抗癌中存在协同作用。表阿霉素广泛应用于膀胱癌的辅助化疗中。在这里,我们假设膀胱癌中死亡受体 DR4和 DR5表达水平与膀胱癌患者的术后化疗及预后相关。方法采用免疫组化法检测229例经尿道切除术后的膀胱癌患者及癌旁正常组织中的DR4和DR5的表达。结果膀胱癌患者细胞质中 DR4和 DR5的表达率分别为75.1％和74．2％。DR4与 DR5均高表达的患者术后10年随访期间的无复发生存率明显好于低表达者。多变量分析显示 DR4（P＜0．001）、DR5（P＜0．001）的表达及表阿霉素的治疗（P＝0．034）是膀胱癌患者预后的独立指标,而且表阿霉素治疗能够明显提高DR4（P＝0．006）或DR5（P＝0．042）高表达的膀胱癌患者的无复发生存率。结论 DR4和DR5均高表达的患者可能更适合接受表阿霉素治疗。